Ligand binding assay
Ligand Binding Assays (LBA) is a term that refers to an assay, or an analytic procedure, whose procedure or method relies of the binding of ligand molecules to receptors.[1] There is then a detection method used to determine the presence and extent of the ligand-receptor complexes formed, and this is usually determined electrochemically or through a fluourescence method.[2] This type of analytic test can be used to test for the presence of target molecules in a sample, that are known to bind to the receptor.[3] It can also be used as a diagnostic tool (ELISA).
There are numerous types of ligand binding assays, both radioactive and non-radioactive.[4][5][6] As such, ligand binding assays are superset of radiobinding assays, which are the conceptual inverse of radioimmunoassays (RIA). Some newer types are called "mix-and-measure" assays because they do not require separation of bound from free ligand.[5]
History
Historically, LBA techniques were used extensively to quantify hormone or hormone receptor concentrations in plasma or in tissue. The ligand-binding assay methodology quantified the concentration of the hormone in the test material by comparing the effects of the test sample to the results of varying amounts of known protein (the ligand).
A prominent use was the test for classification of estrogen receptor status (ER) and progesterone receptor (PR) in breast tumors. The LBA was performed by homogenization of fresh or frozen tumor tissue, incubation of centrifuged cytosol with increasing quantities of radioactive estradiol-17β, followed by separation of unbound estradiol with dextran-coated charcoal (DCC) to quantify the concentration of high-affinity estrogen-binding sites (ER) using standard amounts of known protein.[7] It was replaced by immunohistochemistry (IHC), which may be superior to LBA for assessing estrogen receptor (ER) status in primary breast cancer because it is easier, safer, and less expensive, and has an equivalent or better ability to predict response to adjuvant endocrine therapy.[8][9]
Once accurate and reliable models of ligands and corresponding bindings sites have been investigated, researchers place them in the Protein Data Bank (PDB), through which future researchers can obtain such structures.[10]
Applications for Ligand Binding Assays
Binding assays provide a measure of the interactions that occur between two molecules, such as protein-bindings, as well as the degree of affinity (weak, strong, or no connection) for which the reactants bind together.[11] Essential aspects of binding assays include, but are not limited to, the concentration level of reactants or products (see radioactive section), maintaining the equilibrium constant of reactants throughout the assay, and the reliability and validity of linked reactions.[11] Although binding assays are simple, they fail to provide information on whether or not the compound being tested affects the target's function.[12]
Radioactive
Levels of radioactivity for a radioligand (per mole) is known as the specific activity (SA), which is known as Ci/mmol.[13] The actual concentration of a radioligand is determined by the specific stock mix for which the radioligand originated (from the manufactures.)[13] The following equation determines the actual concentration:
Commercially purchased ligands with radioactive properties:
- Manufacturer's should label each ligand with its individual radioactivity measure, chemical purity and storage suggestions for scientists' use.[14]
- Ligands obtained in this fashion are advised to remain at room temperature (in order to equilibriate) prior to exposure to air, as this exposure may very well cause condensation due to water vapor build-up.[14]
Radioligands should ideally have high affinity, low non-specific binding, high specific activity to detect low receptor densities, and receptor specificity.[15]
Saturation Binding
Saturation binding analysis can determine receptor affinity and density. Saturation analysis requires that the concentration chosen must be determined empirically for a new ligand. There are two common strategies that are adopted for this type of experiment:[15] Increasing the amount of radioligand added while maintaining a constant specific activity[15] and maintaining a constant concentration of radioligand and decreasing the specific activity of the radioligand (by addition of unlabeled ligand).[15]
Scatchard Plot
The use of a Scatchard plot can be used to show radioligand affinity. Here is an example of a Scatchard plot:
In the plots, the ratio of Bound/Free radioligand is plotted against the Bound radioligand. The slope of the line is equal to the negative reciprocal of the affinity constant (K). The intercept of the line with the X axis is an estimate of Bmax.[15] The Scatchard plot can be standardized against an appropriate reference so that there can be a direct comparison of receptor density in different studies and tissues.[15] This sample plot indicates that the radioligand binds with a single affinity. If the ligand were to have bound to multiple sites that have differing radioligand affinities, then the Scatchard plot would have shown a concave line instead.[15]
Nonlinear Curve Fitting Programs
Nonlinear curve fitting programs, such as Equilibrium Binding Data Analysis (EBDA) and LIGAND, are used to calculate estimates of binding parameters from saturation and competition-binding experiments.[16] EBDA performs the initial analysis, which converts measured radioactivity into molar concentrations and creates Hill slopes and Scatchard transformations from the data. The analysis made by EBDA can then be used by LIGAND to estimate a specified model for the binding.[16]
Competition Binding
Competition Binding is used to determine the presence of selectivity for a particular ligand for receptor sub-types, which allows the determination of the density and proportion of each sub-type in the tissue.[15] Competition curves can be obtained by plotting specific binding as a percentage of the total binding against the log concentration of the competing ligand.[15] A steep competition curve is usually indicative of binding to a single population of receptors, whereas a shallow curve or a curve with clear inflection points is indicative of multiple populations of binding sites.[16]
Non-radioactive
Despite the different techniques used for non-radioactive assays, they require that ligands exhibit similar binding characteristics to its radioactive equivalent. Thus, results in both non-radioactive and radioactive assays will remain consistent. [5] One of the largest differences between radioactive and non-radioactive ligand assays are in regards to dangers to human health. Radioactive assays are harmful in that they produce radioactive waste; whereas, non-radioactive ligand assays utilize a different method to avoid producing toxic waste. These methods include, but are not limited to, fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), and surface plasmon resonance (SPR). In order to measure process of ligand-receptor binding, most non-radioactive methods require that labeling avoids interfering with molecular interactions. [5]
Fluorescence Polarization (FP)
Fluorescence polarization is synonymous with fluorescence anisotropy. This method measures the change in the rotational speed of a fluorescent-labeled ligand once it is bound to the receptor.[5] Polarized light is used in order to excite the ligand, and the amount of light emitted is measured.[5] Depolarization of the emitted light depends on the size of the present ligand.[5] If a small ligand is used, it will have a large depolarization, which will rapidly rotate the light.[5] If the ligand utilized is of a larger size, the resulting depolarization will be reduced.[5] An advantage of this method is that it requires only one labeling step.[5] However, if this method is used at low nanomolar concentrations, results will turn out less precise. [5]
Fluorescence Resonance Energy Transfer (FRET)
Fluorescence Resonance Energy Transfer utilizes energy transferred between the donor and the acceptor molecules that are in close proximity.[5] FRET uses a fluorescence labeled ligand like FP.[5] Energy transfer within FRET begins by exciting the donor.[5] The dipole-dipole interaction between the donor and the acceptor molecule transfers the energy from the donor to the acceptor molecule.[5] If the ligand is bound to the receptor-antibody complex, then the acceptor will emit light.[5] When using FRET, it is critical that there is a distance smaller than 10 nm between the acceptor and donor, in addition to an overlapping absorption spectrum between acceptor and donor, and that the antibody does not interfere or block the ligand binding site.[5]
Surface Plasmon Resonance (SPR)
.jpg?lang=en)
Surface Plasmon Resonance does not require labeling of the ligand.[5] Instead, it works by measuring the change in the angle at which the polarized light is reflected from a surface (refractive index).[5] The angle is related to the change is mass or layer of thickness, such as immobilization of a ligand changing the resonance angle, which increases the reflected light.[5] The device for which SPR is derived includes a sensor chip, a flow cell, a light source, a prism, and a fixed angle position detector.[5]
Liquid Phase Ligand Binding Assays
Real-Time Polymerase Chain Reaction (RT-qPCR)
Real-time polymerase chain reaction.
Immunoprecipitation
The liquid phase ligand binding assay of Immunoprecipitation (IP) is a method that is used to purify or enrich a specific protein or a group of proteins using an antibody from a complex mixture. The extract of disrupted tissue or cells is mixed with an antibody against the antigen of interest, which produces the antigen-antibody complex.[17] When antigen concentration is low, the antigen-antibody complex precipitation can take hours or even days and becomes hard to isolate the small about of precipitate formed.[17] The enzyme-linked immunosorbent assay (ELISA) or Western blotting are two different ways that can be used to analyze the purified antigen(s) obtained. This method also involves any assay that proteins are affinity-purified on a small scale, which uses a binding protein immobilized on a solid support.[18] The immobilized protein complex can be accomplished either in a single step or successively.[18] IP can also be used in conjunction with biosynthetic radioisotope labeling. Using this technique combination, one can determine if a specific antigen is synthesized by a tissue or by a cell.[17]
Solid Phase Ligand Binding Assays
Multiwell Plate Assays
Multiwell plates are multiple petri dishes incorporated into one container, with the number of individual wells ranging from 6 to over 1536. Multiwell Plate Assays are convenient for handling necessary dosages and replicates.[19] There are a wide range of plate types that have a standardized footprint, supporting equipment, and measurement systems.[19] Electrodes can be integrated into the bottom of the plates as a solid phase to capture support for the binding assays.[20] The binding reagents become immobilized on the electrode surface and then can be analyzed.[20]
On-Bead Ligand Binding assays
On-Column Ligand Binding Assays
Filter Assays
This solid phase ligand binding assay uses filters to measure the affinity between two molecules. In a filter binding assay, the filters are used to trap cell membranes by sucking the medium through them.[14] This rapid method occurs at a fast speed in which filtration and a recovery can be achieved for the found fraction.[21] Washing filters with a buffer removes residual unbound ligands and any other ligands present that are capable of being washed away from the binding sites.[14] The receptor-ligand complexes present while the filter is being washed will not dissociate significantly because they will be completely trapped by the filters.[14] It is recommended to use an ice-cold wash buffer.[14] Characteristics of the filter are important for each job being done. A thicker filter is useful to get a more complete recovery of small membrane pieces, but may require a longer wash time.[14] It is recommended to pretreat the filters to help trap negatively charged membrane pieces.[14] Soaking the filter in a solution that would give the filter a positive surface charge would attract the negatively charged membrane fragments.[14]
Selectivity and Specificity
Drug effects are a result of their Binding selectivity with Macromolecule properties of an organism, or the affinity with which different ligands bind to a substrate.[22] More specifically, the specificity and selectivity of a Ligand (biochemistry) to its respective receptor provides researchers the opportunity to isolate and produce specific drug effects through the manipulation of ligand concentrations and receptor densities.[22] Hormones and neurotransmitters are essential endogenous regulatory ligands that affect physiological receptors within an organism.[22] Drugs that act upon these receptors are incredibly selective in order to produce required responses from signaling molecules.[22]
Specific binding types to ligand and receptor interactions: [22]
| Mimics Endogenous Effects | Inhibits Endogenous Effects |
|---|---|
| Agonist | Antagonist |
| Partial Agonist | Negative Antagonists (see: Inverse agonist) |
Technological Advances
A prominent signaling molecule in cells is Ca++, which can be detected with a Fluo-4 acetoxymethyl dye. It binds to free Ca++ ions, which in turn slightly increase fluorescence of the Fluo-4 AM.[16] The drawback of the Fluo-4 dye formulation is that a washing step is required to remove extracellular dye, which may provide unwanted background signals. Washing puts stress on the cells and consumes time, which inhibits speedy analysis.[16] Recently, an alternative dye solution and microplate system has been developed called FLIPR® (fluorometric imaging plate reader), which uses a Calcium 3 assay reagent that does not require a washing step. As a result, change in dye fluorescence can be viewed in real time with no delay using an excitatory laser and a charge-coupled device.[16]
Many other ligand binding assays require a filtration step to separate bound and unbound ligands before screening. A method called Scintillation proximity assay (SPA) has been recently developed, which eliminates this otherwise crucial step. It works through crystal lattice beads which are coated with ligand coupling molecules and filled with cerium ions. These give off bursts of light when stimulated by an isotope, which can easily be measured. Ligands are radiolabeled using either 3H or 125I, and released into the assay. Since only the radioligands that directly bind to the beads initiate a signal, free-ligands do not interfere during the screening process.[16]
Limitations
Through the use of The Validation Helper for LIgands and Binding Sites (VHELIBS),[10] researchers are learning how to gain a better sense (and validation) of ligands and their binding sites. Specifically, while the use of Crystallography provides detailed models of ligands and corresponding binding sites, this technique may very well produce incorrect models between or within the same model.[10] As a result of such flaws, ligands and binding sites are invalid, and the level of molecular fitness of a ligand and binding site used for the creation of drugs, for example, are jeopardized.[10]
By nature, assays must be carried out in a controlled environment in vitro, so this method does not provide information abound receptor binding in vivo. The results obtained can only verify that a specific ligand fits a receptor, but assays provide no way of knowing the distribution of ligand-binding receptors in an organism. In vivo ligand binding and receptor distribution can be studied using Positron Emission Tomography (PET), which works by induction of a radionuclide into a ligand, which is then released into the body of a studied organism. The radiolabeled ligands are spatially located by a PET scanner to reveal areas in the organism with high concentrations of receptors.[16]
See also
References
- ^ Luckey, J.; et al. (1993). Methods in Enzymology. 218 (12): 154–172.
{{cite journal}}: Explicit use of et al. in:|last=(help); Missing or empty|title=(help) - ^ Ninfa; et al. (2010). Fundamental Laboratory Approaches for Biochemistry and Biotechnology. Dearborn, MI: University of Michigan.
{{cite book}}: Explicit use of et al. in:|last=(help) - ^ Mullis, K.B, and Faloona, F.L. (1987). Methods in Enzymology. 155 (21): 335–350.
{{cite journal}}: Missing or empty|title=(help)CS1 maint: multiple names: authors list (link) - ^ Attention: This template ({{cite pmid}}) is deprecated. To cite the publication identified by PMID 9667878, please use {{cite journal}} with
|pmid=9667878instead. - ^ a b c d e f g h i j k l m n o p q r s t u Attention: This template ({{cite pmid}}) is deprecated. To cite the publication identified by PMID 16253574, please use {{cite journal}} with
|pmid= 16253574instead. - ^ Joseph R. Lakowicz. (1991) Topics in Fluorescence Spectroscopy: Biochemical applications.
- ^ Attention: This template ({{cite pmid}}) is deprecated. To cite the publication identified by PMID 4345203, please use {{cite journal}} with
|pmid=4345203instead. - ^ Attention: This template ({{cite pmid}}) is deprecated. To cite the publication identified by PMID 10334533, please use {{cite journal}} with
|pmid=10334533instead. - ^ Attention: This template ({{cite pmid}}) is deprecated. To cite the publication identified by PMID 16491599, please use {{cite journal}} with
|pmid=16491599instead. - ^ a b c d Cereto-Massagué, Adrià (201). "The good, the bad and the dubious: VHELIBS, a validation helper for ligands and binding sites". Journal of Cheminformatics. 5 (36): 1–9. doi:10.1186/1758-2946-5-36.
{{cite journal}}: Unknown parameter|coauthors=ignored (|author=suggested) (help)CS1 maint: unflagged free DOI (link) - ^ a b POLLARD, Thomas D. (1 December 2010). "A Guide to Simple and Informative Binding Assays". Molecular Biology of the Cell. 21: 4061–4067.
- ^ Offermanns, Stefan (2008). Encyclopedia of molecular pharmacology (2nd ed. ed.). Berlin: Springer. p. 585. ISBN 9783540389163.
{{cite book}}:|edition=has extra text (help); Unknown parameter|coauthors=ignored (|author=suggested) (help) - ^ a b c Kahl, Steven D. (1 May 2012). "Calculations and Instrumentation used for Radioligand Binding Assays". Assay Guidance Manual: 1–21.
{{cite journal}}:|access-date=requires|url=(help); Unknown parameter|coauthors=ignored (|author=suggested) (help) - ^ a b c d e f g h i Hulme, Edward C. (November 2010). "Ligand binding assays at equilibrium: validation and interpretation". British Journal of Pharmacology. 161 (6): 1219–1237. doi:10.1111/j.1476-5381.2009.00604.x.
{{cite journal}}: Unknown parameter|coauthors=ignored (|author=suggested) (help) - ^ a b c d e f g h i Anthony P. Davenport and Fraser D. Russel (1996). "Radioligand Binding Assays: Theory and Practice". In Stephen J. Mather (ed.). Current directions in radiopharmaceutical research and development. Springer Netherlands. pp. 169–179. ISBN 978-94-010-7289-2.
- ^ a b c d e f g h Davenport, Anthony P. (2005). Receptor Binding Techniques. Humana Press. pp. 18–19, 101–102, 121–122, 203–204. ISBN 1-58829-420-X.
- ^ a b c Goldsby, Richard A. (2003). Immunology (5e éd. ed.). New York: W. H. Freeman. p. 152. ISBN 0716749475.
- ^ a b "Immunoprecipitation (IP) technical guide and protocols" (PDF). Thermo Fisher Scientific Inc. Retrieved 20 March 2014.
- ^ a b Robinson, C. Jane (January 2014). "Assay Acceptance Criteria for Multiwell-Plate–Based Biological Potency Assays". BioProcess International. 12 (1): 30–41.
{{cite journal}}: Unknown parameter|coauthors=ignored (|author=suggested) (help) - ^ a b Masood N. Khan, John W. Findlay, ed. (2009). Ligand-binding assays development, validation, and implementation in the drug development arena. Hoboken, N.J.: John Wiley & Sons. ISBN 0470541490.
- ^ Moss, ed. by Tom (2001). "Filter-Binding Assays". DNA-protein interactions : principles and protocols (2nd ed.). Totowa, NJ: Humana Press. pp. 1–12. ISBN 9780896036710.
{{cite book}}:|first=has generic name (help) - ^ a b c d e Goodman & Gilman's The Pharmacological Basis of Therapeutics. The McGraw Hill Companies Inc. 1996. pp. 29–37. ISBN 0-07-026266-7.